Cleft lip is one of most common birth defects. Caused by disruption of normal craniofacial development, cleft lip affects approximately 1 in 500 newborns worldwide. The long term goal of this proposed research is to understand the mechanisms of upper lip development and of orofacial cleft pathogenesis. Mutations of Platelet Derived Growth Factor Receptor (PDGFR) signaling have been tightly linked to cleft lip/palate in humans and mice, suggesting an evolutionarily conserved role in craniofacial development. During embryo development, the upper lip is formed by fusion of medial nasal process (MNP) and maxillary processes, both originating from neural crest cells. This proposed research is aimed at characterizing the role of PDGFR in development of MNP and of neural crest cells during upper lip morphogenesis. First, we will generate a novel transgenic line Alx3Cre to examine the role of PDGFR specifically in MNP development. Cell proliferation and cell survival will be assayed in the MNP of conditional knockout and control embryos using immunohistochemistry methods. Scratch assay and transwell assay will be carried out to analyze the role of PDGFR signaling in MNP cell migration. In the second specific aim, we will examine the role of PDGFR specifically in neural crest cells. Lineage tracing method and in vitro explants culture approaches will be combined with immunohistochemistry experiments in the conditional knockout and control embryos. In the third specific aim, we will analyze the role of PDGFR engaged PI3K signaling in MNP and neural crest development using PDGFR signaling mutant PDGFR PI3K/PI3K mice. In vitro cell migration assays and in vivo mouse genetic studies proposed in first and second specific aims will be carried out on PDGFR PI3K/PI3K and control embryos. Results of the proposed works will reveal the fundamental role of PDGFR in MNP and neural crest cell development during upper lip morphogenesis. The results of proposed research will provide novel information to understand the fundamental mechanisms of MNP and upper lip formation. This study will benefit treatment and prevention of cleft lip in the future, to ultimately reduce the occurrence of this birth defect in newborns.